Alkaline proteases are one of the most important group of enzymes that are indispensable in a number of different industrial sectors. In this work, the effect of copper ions (Cu2+) was investigated for improving the thermostability and hydrolytic performance of Bacillus clausii GMBE 42 alkaline protease at different temperatures (45–65°C). Maximal residual activity was observed in the presence of 5 mM CuCl2. The enzyme was thermoinactivated according to first‐order kinetics. A stabilization effect caused by copper ions was the result of a decrease in both autolysis and thermoinactivation rates. Thermodynamic analysis of the thermoinactivation process showed that Ea,i, ΔGi, and ΔHi values of the enzyme were higher in the presence of copper ions, but there was no measurable change in ΔSi values. These results show the thermostabilizing potential of copper ions on the enzyme. Lower Km values and higher kcat and kcat/Km values were obtained in the presence of copper ions, which is an indication of the nonessential activation of the enzyme by copper ions. Thermodynamic analysis of casein hydrolysis showed that in presence of copper ions Ea, ΔG≠, ΔH≠, ΔnormalGnormalE−normalS≠, and ΔnormalGnormalE−normalT≠ values of enzyme were lower, but there was no change in ΔS≠ values. This is so far the first study that investigates the effect of cations on the basic catalytic and thermodynamic properties of an alkaline serine protease, which may be used to remove protein wastes from various industries such as food and leather processing.

An alkali tolerant Bacillus strain having extracellular serine alkaline protease activity was newly isolated from compost and identified as Bacillus clausii GMBE 22. An alkaline protease (AP22) was 4.66-fold purified in 51.5% yield from Bacillus clausii GMBE 22 by ethanol precipitation and DEAE-cellulose anion exchange chromatography. The purified enzyme was identified as serine protease by LC-ESI-MS analysis. Its complete inhibition by phenylmethanesulfonylfluoride (PMSF) also justified that it is a serine alkaline protease. The molecular weight of the enzyme is 25.4 kDa. Optimal temperature and pH values are 60 degrees C and 12.0, respectively. The enzyme showed highest specificity to N-Suc-Ala-Ala-Pro-Phe-pNA. The K(m) and k(cat) values for hydrolysis of this substrate are 0.347 mM and 1141 min(-1) respectively. The enzyme was affected by surface active agents to varying extents. The enzyme is stable for 2 h at 30 degrees C and pH 10.5. AP22 is also stable for 5 days over the pH range 9.0-11.0 at room temperature. AP22 has good pH stability compared with the alkaline proteases belonging to other strains of Bacillus clausii reported in the literature.

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